Genome wide DNA copy number analysis in cholangiocarcinoma using high resolution molecular inversion probe single nucleotide polymorphism assay.

In order to study molecular similarities and differences of intrahepatic (IH-CCA) and extrahepatic (EH-CCA) cholangiocarcinoma, 24 FFPE tumor samples (13 IH-CCA, 11 EH-CCA) were analyzed for whole genome copy number variations (CNVs) using a new high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay. Common in both tumor subtypes the most frequent losses were detected on chromosome 1p, 3p, 6q and 9 while gains were mostly seen in 1q, 8q as well as complete chromosome 17 and 20. Applying the statistical GISTIC (Genomic Identification of Significant Targets in Cancer) tool we identified potential novel candidate tumor suppressor- (DBC1, FHIT, PPP2R2A) and oncogenes (LYN, FGF19, GRB7, PTPN1) within these regions of chromosomal instability. Next to common aberrations in IH-CCA and EH-CCA, we additionally found significant differences in copy number variations on chromosome 3 and 14. Moreover, due to the fact that mutations in the Isocitrate dehydrogenase (IDH-1 and IDH-2) genes are more frequent in our IH-CCA than in our EH-CCA samples, we suggest that the tumor subtypes have a different molecular profile. In conclusion, new possible target genes within regions of high significant copy number aberrations were detected using a high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay, which opens a future perspective of fast routine copy number and marker gene identification for gene targeted therapy.

Hedgehog pathway as a potential treatment target in human cholangiocarcinoma.

BACKGROUND: Innovative treatment concepts targeting essential signaling pathways may offer new chances for patients suffering from cholangiocarcinoma (CCC). For that, we performed a systematic molecular genetic analysis concerning the Hedgehog activity in human CCC samples and analyzed the effect of Hh inhibition on CCC cells in vitro and in vivo. METHODS: Activation of the Hh pathway was analyzed in 50 human CCC samples using quantitative polymerase chain reaction (qPCR). The efficacy of Hh inhibition using cyclopamine and BMS-833923 was evaluated in vitro. In addition, the effect of BMS-833923, alone or in combination with gemcitabine, was analyzed in vivo in a murine subcutaneous xenograft model. RESULTS: Expression analysis revealed a significant activation of the Hh-signaling pathway in nearly 50% of CCCs. Hh inhibition resulted in a significant decrease in cell proliferation of CCC cells. Moreover, a distinct inhibition of tumor growth could be seen as a result of a combined therapy with BMS-833923 and gemcitabine in CCC xenografts. CONCLUSION: The results of our study suggest that the Hh pathway plays a relevant role at least in a subset of human CCC. Inhibition of this pathway may represent a possible treatment option for CCC patients in which the Hh pathway is activated.

Impaired Pten expression in human malignant peripheral nerve sheath tumours.

Malignant peripheral nerve sheath tumours (MPNST) are aggressive sarcomas that develop in about 10% of patients with the genetic disease neurofibromatosis type 1 (NF1). Molecular alterations contributing to MPNST formation have only partially been resolved. Here we examined the role of Pten, a key regulator of the Pi3k/Akt/mTOR pathway, in human MPNST and benign neurofibromas. Immunohistochemistry showed that Pten expression was significantly lower in MPNST (n=16) than in neurofibromas (n=16) and normal nervous tissue. To elucidate potential mechanisms for Pten down-regulation or Akt/mTOR activation in MPNST we performed further experiments. Mutation analysis revealed absence of somatic mutations in PTEN (n=31) and PIK3CA (n=38). However, we found frequent PTEN promotor methylation in primary MPNST (11/26) and MPNST cell lines (7/8) but not in benign nerve sheath tumours. PTEN methylation was significantly associated with early metastasis. Moreover, we detected an inverse correlation of Pten-regulating miR-21 and Pten protein levels in MPNST cell lines. The examination of NF1-/- and NF1+/+Schwann cells and fibroblasts showed that Pten expression is not regulated by NF1. To determine the significance of Pten status for treatment with the mTOR inhibitor rapamycin we treated 5 MPNST cell lines with rapamycin. All cell lines were sensitive to rapamycin without a significant correlation to Pten levels. When rapamycin was combined with simvastatin a synergistic anti-proliferative effect was achieved. Taken together we show frequent loss/reduction of Pten expression in MPNST and provide evidence for the involvement of multiple Pten regulating mechanisms.